Neoantigens from tumor cells are peptide fragments of mutated proteins that contain a mutation, and are capable of being presented in the cleft of Major Histocompatibility Complex (MHC) Class I molecules on the surfaces of cells within the tumor, where they can be surveyed by CD8-positive T cells. The tumor-specificity of neoantigens, coupled with the ability of neoantigen-specific T cells to specifically kill cancer cells, has made tumor neoantigens increasingly important for cancer immunotherapy. Additionally, T cell receptors (TCRs) that recognize specific neoantigens are candidates for TCR-engineered cell based therapies for targeting infections that produce neoantigens.
Putative neoantigens have been previously identified by tumor exome analysis, and subsequently rank-ordered according to their MHC binding strength using in silico analysis. However, finding which candidate neoantigens are actually promoting T cell tumor infiltration is challenging for several reasons. First, a patient tumor that has a mutation density of 10 per 1 million expressed DNA base pairs might be predicted to have on the order of 50 putative neoantigens that exhibit a binding constant (Kd) to a given human leukocyte antigen (HLA) genotype MHC of 500 nM or lower. Furthermore, any particular neoantigen-specific T cell population is likely to exist in very low abundance in a patient, rendering isolation and/or identification of neoantigen-specific T cells very difficult. Related challenges to these issues include the pairing of neoantigens to their cognate TCRs. Nevertheless, these neoantigen-T cell pairing interactions are at the core of cancer immunotherapy, and so there has been significant effort towards meeting these challenges. Previous approaches for neoantigen-specific T cell pairing have shown to be laborious, non-quantitative, and/or they may only identify one or two T cell populations per HLA genotype due to limited sensitivity.